CN111518696A - Preparation method and application of microbial soil remediation agent - Google Patents
Preparation method and application of microbial soil remediation agent Download PDFInfo
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- CN111518696A CN111518696A CN202010185247.0A CN202010185247A CN111518696A CN 111518696 A CN111518696 A CN 111518696A CN 202010185247 A CN202010185247 A CN 202010185247A CN 111518696 A CN111518696 A CN 111518696A
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- 239000002689 soil Substances 0.000 title claims abstract description 132
- 238000005067 remediation Methods 0.000 title claims abstract description 52
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 50
- 230000000813 microbial effect Effects 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 229910052793 cadmium Inorganic materials 0.000 claims abstract description 52
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000007788 liquid Substances 0.000 claims abstract description 39
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 230000001580 bacterial effect Effects 0.000 claims abstract description 25
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 21
- 239000003223 protective agent Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 12
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 12
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000008223 sterile water Substances 0.000 claims abstract description 12
- 239000000230 xanthan gum Substances 0.000 claims abstract description 12
- 229920001285 xanthan gum Polymers 0.000 claims abstract description 12
- 229940082509 xanthan gum Drugs 0.000 claims abstract description 12
- 235000010493 xanthan gum Nutrition 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 4
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000011081 inoculation Methods 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 4
- 238000005303 weighing Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 35
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000012216 screening Methods 0.000 claims description 9
- 239000002344 surface layer Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000009630 liquid culture Methods 0.000 claims description 8
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 8
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 8
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 8
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 8
- 235000011046 triammonium citrate Nutrition 0.000 claims description 8
- 239000001393 triammonium citrate Substances 0.000 claims description 8
- YKYOUMDCQGMQQO-UHFFFAOYSA-L cadmium dichloride Chemical compound Cl[Cd]Cl YKYOUMDCQGMQQO-UHFFFAOYSA-L 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 239000000575 pesticide Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 230000035699 permeability Effects 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 235000011187 glycerol Nutrition 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- DALUDRGQOYMVLD-UHFFFAOYSA-N iron manganese Chemical compound [Mn].[Fe] DALUDRGQOYMVLD-UHFFFAOYSA-N 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002680 soil gas Substances 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002912 waste gas Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
Abstract
The invention relates to the technical field of heavy metal pollution treatment, and particularly discloses a preparation method of a microbial soil remediation agent, which comprises the steps of selecting a high-cadmium-resistant bacterial strain QN-3 separated from heavy metal polluted soil, and preparing a seed solution through enlarged culture; under the aseptic condition, inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10%, and carrying out liquid seed amplification fermentation culture for 3-5 days to prepare liquid bacterial liquid; weighing Tween-20, xanthan gum, glycerol, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, mixing well, adding sterile water to constant volume, and making into protectant; adding a protective agent into liquid bacterial liquid of the high cadmium-resistant strain QN-3, and uniformly mixing to prepare the microbial soil restoration agent, wherein the content of effective viable bacteria of the high cadmium-resistant strain QN-3 in the microbial soil restoration agent is not lower than 2 hundred million/ml. The invention overcomes the defects of the prior art, and can improve the soil environment and effectively reduce the cadmium content in the soil by preparing the soil remediation agent through the viable bacteria of the high cadmium-resistant bacterial strain.
Description
Technical Field
The invention relates to the technical field of heavy metal pollution treatment, and particularly belongs to a preparation method and application of a microbial soil remediation agent.
Background
The soil is a loose surface layer which has fertility and can grow plants on the surface of the land, the thickness of the soil is about 2m generally, and the soil not only provides mechanical support capability for the growth of the plants, but also provides fertility factors such as water, fertilizer, gas, heat and the like required by the growth and development of the plants. In recent years, as the population is rapidly increased and the industry is rapidly developed, solid waste is continuously piled and dumped to the surface of soil, harmful waste water is continuously permeated into the soil, and waste gas discharged by automobiles, harmful gas and floating dust in the atmosphere are continuously fallen in the soil along with rainwater. The improvement of the level of agricultural chemistry causes a large amount of chemical fertilizers and pesticides to scatter into the environment, so that the probability that the soil is polluted by non-point sources is increased, the degree of the pollution is increased, and the polluted area is enlarged continuously under the influence of water and soil loss, wind erosion and the like. When the harmful substances in the soil are excessive and exceed the self-cleaning capacity of the soil, the composition, the structure and the function of the soil are changed, the microbial activity is inhibited, the harmful substances or decomposition products thereof are gradually accumulated in the soil and absorbed by the human body through a biological circulation way, and the degree harmful to the health of the human body is reached, namely the soil pollution.
The remediation of heavy metal contaminated soil is mainly based on 2 strategies: firstly, removing heavy metals from soil to achieve the aim of cleaning the soil; and secondly, the heavy metal is immobilized in the soil to limit the release of the heavy metal, so that the risk of the heavy metal is reduced. In recent years, research on remediation technologies for heavy metal contaminated soil has been greatly developed, and the remediation technologies are mainly classified into physical/chemical remediation, biological remediation and agricultural ecological remediation according to process principles.
In recent years, researches on methods for repairing soil by using microorganisms are more and more, but the method for repairing soil polluted by heavy metals by using a microorganism live bacteria preparation produced by a fermentation engineering technology is rarely reported at home.
Disclosure of Invention
The invention aims to provide a preparation method and application of a microbial soil remediation agent, overcomes the defects of the prior art, prepares the soil remediation agent by using viable bacteria of a high cadmium-resistant bacterial strain, and can improve the soil environment and effectively reduce the cadmium content in the soil.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a preparation method of a microbial soil remediation agent comprises the following process steps:
(1) selecting a high cadmium-resistant strain QN-3 separated from heavy metal contaminated soil, and preparing a seed solution through enlarged culture;
(2) under the aseptic condition, inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10%, and carrying out liquid seed amplification fermentation culture at 28-32 ℃ for 3-5d to prepare liquid bacterial liquid;
(3) weighing 0.05-0.3% w/v of tween-20, 0.1-0.5% w/v of xanthan gum, 1-5% w/v of glycerol, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, mixing uniformly, adding sterile water to a constant volume, and preparing the protective agent;
(4) in the liquid bacterial liquid of the high cadmium-resistant strain QN-3, according to the following liquid bacterial liquid: adding the protective agent according to the volume ratio of 0.5-1:1 of the protective agent, and uniformly mixing to prepare the microbial soil remediation agent, wherein the content of effective viable bacteria of a high cadmium-resistant strain QN-3 in the microbial soil remediation agent is not lower than 2 hundred million/ml.
Further, the screening method of the high cadmium-resistant strain QN-3 comprises the following steps: selecting a plurality of heavy metal contaminated soil samples to prepare soil suspension, taking supernatant liquid to be evenly coated on a solid peptone culture medium containing 100mg/L cadmium chloride to respectively pick single bacterial colonies, adopting a plate streaking separation method to respectively seed the bacterial colonies to solid culture media with cadmium concentrations of 100, 150 and 200mg/L, then screening out high cadmium-resistant strains with strong cadmium resistance, continuously purifying the single bacterial colonies for 4 times by adopting a plate streaking separation method, sequentially inoculating the strains obtained by purification to the same screening culture medium containing a series of concentration gradients of 250 and 24000mg/L of cadmium concentrations, respectively, culturing at room temperature, and selecting the strains resisting high cadmium to obtain the high cadmium-resistant strains QN-3.
Further, the high cadmium-tolerant strain QN-3 is deposited in Guangdong province microbial culture collection center (GDMCC) in 2018, 05 and 28 months, and the strain deposit number is GDMCC No: 60378.
further, the liquid culture medium in the step (2) is: 27-33g of peptone, 9-11g of yeast powder, 27-33g of glucose, 1.1mL of tween-800.9, 1.8-2.2g of triammonium citrate, 0.6-0.8g of magnesium sulfate heptahydrate, 0.045-0.055g of manganese sulfate monohydrate and 9.0-11.0g of calcium carbonate; adjusting the pH value to 6.9-7.3.
Further, the protective agent in the step (3) comprises the following components in parts by weight: 240 parts of tween-20200, 1-8 parts of xanthan gum, 4-10 parts of glycerol, 0.2-0.8 part of disodium hydrogen phosphate, 0.1-0.5 part of potassium dihydrogen phosphate and magnesium sulfate, and the balance of sterile water.
According to the preparation method and the application of the microbial soil remediation agent, the application of the microbial soil remediation agent comprises the following application methods:
A. ploughing the soil with the surface layer thickness of 10-18cm of cadmium-polluted soil by using a rotary cultivator to keep the air permeability of the soil;
B. watering and irrigating to keep the soil moisture at 60-80%, selecting the environment temperature at 10-30 ℃, taking sun-shading measures, and mixing the microbial soil remediation agent with water according to the proportion of 1: diluting by 100 mass ratio, and uniformly spraying the diluted microbial soil remediation agent into soil according to the dosage of 30-60L/mu;
C. and (4) after standing for 30 days, repeating the operation of the step B to carry out secondary pesticide spraying, wherein the using amount and the using method of the microbial soil remediation agent are the same as those in the step B, and standing for 30 days.
Further, before the plowing in the step A, a grid multi-point method is adopted for soil sample collection, and then the cadmium content of the collected soil sample is measured.
Compared with the prior art, the invention has the following implementation effects:
according to the preparation method and the application of the microbial soil remediation agent, the soil remediation agent is prepared by the viable bacteria of the high cadmium-resistant bacterial strain, so that the soil environment can be improved, and the cadmium content in the soil can be effectively reduced; the protective agent is added into the microbial inoculum, so that effective nutrient elements can be provided for microorganisms, and the repaired soil can be used as agricultural cultivated land.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples, and any modification is within the scope of the present invention without departing from the spirit of the present invention.
A preparation method of a microbial soil remediation agent comprises the following process steps:
(1) selecting a high cadmium-resistant strain QN-3 separated from heavy metal contaminated soil, and preparing a seed solution through enlarged culture;
(2) under the aseptic condition, inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10%, and carrying out liquid seed amplification fermentation culture at 28-32 ℃ for 3-5 days to prepare liquid bacterial liquid;
(3) weighing 0.05-0.3% w/v of tween-20, 0.1-0.5% w/v of xanthan gum, 1-5% w/v of glycerol, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, mixing uniformly, adding sterile water to a constant volume, and preparing the protective agent;
(4) in the liquid bacterial liquid of the high cadmium-resistant strain QN-3, according to the following liquid bacterial liquid: adding the protective agent according to the volume ratio of 0.5-1:1 of the protective agent, uniformly mixing to prepare the microbial soil restoration agent, wherein the content of effective viable bacteria of a high cadmium-resistant strain QN-3 in the microbial soil restoration agent is not lower than 2 hundred million/ml.
The screening method of the high cadmium-resistant strain QN-3 comprises the following steps: selecting a plurality of heavy metal contaminated soil samples to prepare soil suspension, taking supernatant liquid to be evenly coated on a solid peptone culture medium containing 100mg/L cadmium chloride to respectively pick single bacterial colonies, adopting a plate streaking separation method to respectively seed the bacterial colonies to solid culture media with cadmium concentrations of 100, 150 and 200mg/L, then screening out high cadmium-resistant strains with strong cadmium resistance, continuously purifying the single bacterial colonies for 4 times by adopting a plate streaking separation method, sequentially inoculating the strains obtained by purification to the same screening culture medium containing a series of concentration gradients of 250 and 24000mg/L of cadmium concentrations, respectively, culturing at room temperature, and selecting the strains resisting high cadmium to obtain the high cadmium-resistant strains QN-3.
The high cadmium-resistant strain QN-3 is preserved in Guangdong province microorganism culture collection center (GDMCC) in 2018, 05 and 28 months, and the strain preservation number is GDMCC No: 60378, proposed taxonomy name: cystobasididitun oligophagu, deposit organization address: experimental building 5 of 100 # large college of junior furious district of Guangzhou city, Guangdong province.
The liquid culture medium in the step (2) is as follows: 27-33g of peptone, 9-11g of yeast powder, 27-33g of glucose, 1.1mL of tween-800.9, 1.8-2.2g of triammonium citrate, 0.6-0.8g of magnesium sulfate heptahydrate, 0.045-0.055g of manganese sulfate monohydrate and 9.0-11.0g of calcium carbonate; adjusting the pH value to 6.9-7.3.
The protective agent in the step (3) comprises the following components in parts by weight: 240 parts of tween-20200, 1-8 parts of xanthan gum, 4-10 parts of glycerol, 0.2-0.8 part of disodium hydrogen phosphate, 0.1-0.5 part of potassium dihydrogen phosphate and magnesium sulfate, and the balance of sterile water.
According to the preparation method and the application of the microbial soil remediation agent, the application of the microbial soil remediation agent comprises the following application methods:
A. ploughing the soil with the surface layer thickness of 10-18cm of cadmium-polluted soil by using a rotary cultivator to keep the air permeability of the soil;
B. watering and irrigating to keep the soil moisture at 60-80%, selecting the environment temperature at 10-30 ℃, taking sun-shading measures, and mixing the microbial soil remediation agent with water according to the proportion of 1: diluting by 100 mass ratio, and uniformly spraying the diluted microbial soil remediation agent into soil according to the dosage of 30-60L/mu;
C. and (4) after standing for 30 days, repeating the operation of the step B to carry out secondary pesticide spraying, wherein the using amount and the using method of the microbial soil remediation agent are the same as those in the step B, and standing for 30 days.
Before the plowing in the step A, firstly, a grid multi-point method is adopted for collecting soil samples, and then the cadmium content of the collected soil samples is measured.
Example 1
This example prepares the microbial soil remediation agent according to the method of the present invention, and the liquid culture medium of example 1 is: 27g of peptone, 9g of yeast powder, 27g of glucose, 800.9mL of tween-E, 1.8g of triammonium citrate, 0.6g of magnesium sulfate heptahydrate, 0.045g of manganese sulfate monohydrate and 9.0g of calcium carbonate;
the weight g of each component of the protective agent is as follows: tween-20200 g, xanthan gum 1g, glycerol 4g, disodium hydrogen phosphate 0.2g, potassium dihydrogen phosphate and magnesium sulfate 0.1g, and the balance of sterile water, and the volume is fixed to 1000 ml;
when in use, the rotary cultivator ploughs the soil with the surface layer thickness of 10cm of the cadmium-polluted soil; the diluted microbial soil remediation agent is uniformly sprayed into soil according to the dosage of 30L/mu.
Example 2
This example prepares the microbial soil remediation agent according to the method of the present invention, and the liquid culture medium of example 2 is: 30g of peptone, 10g of yeast powder, 30g of glucose, 801.0mL of tween-E, 2.0g of triammonium citrate, 0.7g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate monohydrate and 10g of calcium carbonate;
the weight g of each component of the protective agent is as follows: tween-20220 g, xanthan gum 4.5g, glycerol 7g, disodium hydrogen phosphate 0.5g, potassium dihydrogen phosphate and magnesium sulfate 0.3g, and sterile water in balance, and keeping the volume to 1000 ml;
when in use, the rotary cultivator ploughs the soil with the surface layer thickness of 10cm of the cadmium-polluted soil; the diluted microbial soil remediation agent is uniformly sprayed into soil according to the dosage of 30L/mu.
Example 3
This example prepares the microbial soil remediation agent according to the method of the present invention, and the liquid culture medium of example 2 is: 33g of peptone, 11g of yeast powder, 33g of glucose, 801.1mL of tween-1, 2.2g of triammonium citrate, 0.8g of magnesium sulfate heptahydrate, 0.055g of manganese sulfate monohydrate and 11g of calcium carbonate;
the protective agent comprises the following components in parts by weight: tween-20240 g, xanthan gum 8g, glycerol 10g, disodium hydrogen phosphate 0.8g, potassium dihydrogen phosphate and magnesium sulfate 0.8g, and the balance of sterile water, and the volume is fixed to 1000 ml;
when in use, the rotary cultivator ploughs the soil with the surface layer thickness of 10cm of the cadmium-polluted soil; the diluted microbial soil remediation agent is uniformly sprayed into soil according to the dosage of 30L/mu.
Example 4
This example prepares the microbial soil remediation agent according to the method of the present invention, and the liquid culture medium of example 1 is: 27g of peptone, 9g of yeast powder, 27g of glucose, 800.9mL of tween-E, 1.8g of triammonium citrate, 0.6g of magnesium sulfate heptahydrate, 0.045g of manganese sulfate monohydrate and 9.0g of calcium carbonate;
the weight g of each component of the protective agent is as follows: tween-20200 g, xanthan gum 1g, glycerol 4g, disodium hydrogen phosphate 0.2g, potassium dihydrogen phosphate and magnesium sulfate 0.1g, and the balance of sterile water, and the volume is fixed to 1000 ml;
when in use, the rotary cultivator ploughs the soil with the surface layer thickness of 14cm of the cadmium-polluted soil; the diluted microbial soil remediation agent is uniformly sprayed into soil according to the dosage of 45L/mu.
Example 5
This example prepares the microbial soil remediation agent according to the method of the present invention, and the liquid culture medium of example 1 is: 27g of peptone, 9g of yeast powder, 27g of glucose, 800.9mL of tween-E, 1.8g of triammonium citrate, 0.6g of magnesium sulfate heptahydrate, 0.045g of manganese sulfate monohydrate and 9.0g of calcium carbonate;
the weight g of each component of the protective agent is as follows: tween-20200 g, xanthan gum 1g, glycerol 4g, disodium hydrogen phosphate 0.2g, potassium dihydrogen phosphate and magnesium sulfate 0.1g, and the balance of sterile water, and the volume is fixed to 1000 ml;
when in use, the rotary cultivator ploughs the soil with the surface layer thickness of 18cm of the cadmium-polluted soil; the diluted microbial soil remediation agent is uniformly sprayed into soil according to the dosage of 60L/mu.
Comparative example 1
Compared with the embodiment 1, the difference of the comparative example is that the liquid bacterial liquid prepared in the preparation method of the microbial soil restoration agent is frozen, dried and crushed to obtain powder, and then the powder is mixed with the protective agent.
Comparative example 2
The comparative example differs from example 1 in that no protective agent was added.
Example of effects:
(1) experimental samples: soil remediation agents prepared according to examples 1 to 5 of the present invention and comparative examples 1 and 2.
(2) The experimental method comprises the following steps: heavy metal contaminated soil: the soil with heavy cadmium pollution of 7 mu is selected from Quanwantoucun of Xuzhou city, Jiangsu province.
(a) Determining the cadmium content of various forms of heavy metal contaminated soil according to detection methods in a standard GB/T23739-2009 atomic absorption method for determining effective lead and cadmium in soil quality and technical specification for monitoring soil environment;
(b) the method comprises the steps of averagely dividing 7 mu of heavy metal contaminated soil into seven groups, respectively applying seven groups of experimental samples to the heavy metal contaminated soil according to a using method, placing and maintaining for 30 days, then applying the samples again, placing and maintaining for 30 days, enabling the seven groups of samples to respectively and fully react with cadmium in the heavy contaminated soil, fully stabilizing, respectively measuring the content of various forms of cadmium in 7 groups of mixed soil, and measuring results are shown in table 1.
TABLE 1 statistics of cadmium morphological changes (mg/kg) of soil before and after remediation
(3) The experimental results are as follows: as can be seen from Table 1, the contents of cadmium in a residual state and cadmium in an iron-manganese state in the heavy metal polluted soil treated by the microbial soil remediation agents prepared in the embodiments 1 to 5 meet the requirements of GB 15618-.
The foregoing is merely exemplary and illustrative of the present inventive concept and various modifications, additions and substitutions of similar embodiments may be made to the specific embodiments described by those skilled in the art without departing from the inventive concept or exceeding the scope of the claims as defined in the accompanying claims.
Claims (7)
1. A preparation method of a microbial soil remediation agent is characterized by comprising the following steps: the method comprises the following process steps:
(1) selecting a high cadmium-resistant strain QN-3 separated from heavy metal contaminated soil, and preparing a seed solution through enlarged culture;
(2) under the aseptic condition, inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10%, and carrying out liquid seed amplification fermentation culture at 28-32 ℃ for 3-5 days to prepare liquid bacterial liquid;
(3) weighing 0.05-0.3% w/v of tween-20, 0.1-0.5% w/v of xanthan gum, 1-5% w/v of glycerol, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, mixing uniformly, adding sterile water to a constant volume, and preparing the protective agent;
(4) in the liquid bacterial liquid of the high cadmium-resistant strain QN-3, according to the following liquid bacterial liquid: adding the protective agent according to the volume ratio of 0.5-1:1 of the protective agent, and uniformly mixing to prepare the microbial soil remediation agent, wherein the content of effective viable bacteria of a high cadmium-resistant strain QN-3 in the microbial soil remediation agent is not lower than 2 hundred million/ml.
2. The method of claim 1, wherein said method comprises the steps of: the screening method of the high cadmium-resistant strain QN-3 comprises the following steps: selecting a plurality of heavy metal contaminated soil samples to prepare soil suspension, taking supernatant liquid to be evenly coated on a solid PDA culture medium containing 100mg/L cadmium chloride, respectively selecting single bacterial colonies, respectively planting the bacterial colonies to solid culture media with cadmium concentrations of 100, 150 and 200mg/L by adopting a plate streaking separation method, then screening out high cadmium-resistant strains with strong cadmium resistance, continuously purifying the single bacterial colonies for 4 times by adopting the plate streaking separation method, sequentially inoculating the strains obtained by purification to the same screening culture medium containing a series of concentration gradients of 250 and 24000mg/L of cadmium concentrations, respectively, culturing at room temperature, and selecting the strains resisting high cadmium to obtain the high cadmium-resistant strains QN-3. .
3. The method of claim 1 or 2, wherein said method comprises the steps of: the high cadmium-resistant strain QN-3 is preserved in Guangdong province microorganism culture collection center (GDMCC) in 2018, 05 and 28 days, and the strain preservation number is GDMCC No: 60378.
4. the method of claim 1, wherein said method comprises the steps of: the liquid culture medium in the step (2) is as follows: 27-33g of peptone, 9-11g of yeast powder, 27-33g of glucose, 1.1mL of tween-800.9, 1.8-2.2g of triammonium citrate, 0.6-0.8g of magnesium sulfate heptahydrate, 0.045-0.055g of manganese sulfate monohydrate and 9.0-11.0g of calcium carbonate; adjusting the pH value to 6.9-7.3.
5. The method of claim 1, wherein said method comprises the steps of: the protective agent in the step (3) comprises the following components in parts by weight: 240 parts of tween-20200, 1-8 parts of xanthan gum, 4-10 parts of glycerol, 0.2-0.8 part of disodium hydrogen phosphate, 0.1-0.5 part of potassium dihydrogen phosphate and magnesium sulfate, and the balance of sterile water.
6. The preparation method and application of the microbial soil remediation agent as claimed in any one of claims 1-5, wherein: the application of the microbial soil remediation agent comprises the following application methods:
A. ploughing the soil with the surface layer thickness of 10-18cm of cadmium-polluted soil by using a rotary cultivator to keep the air permeability of the soil;
B. watering and irrigating to keep the soil moisture at 60-80%, selecting the environment temperature at 10-30 ℃, taking sun-shading measures, and mixing the microbial soil remediation agent with water according to the proportion of 1: diluting by 100 mass ratio, and uniformly spraying the diluted microbial soil remediation agent into soil according to the dosage of 30-60L/mu;
C. and (4) after standing for 30 days, repeating the operation of the step B to carry out secondary pesticide spraying, wherein the using amount and the using method of the microbial soil remediation agent are the same as those in the step B, and standing for 30 days.
7. The preparation method and application of the microbial soil remediation agent of claim 6, wherein the soil remediation agent comprises the following components: before the plowing in the step A, firstly, collecting a soil sample by adopting a grid multipoint method, and then measuring the cadmium content of the collected soil sample.
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